Mark Koylass

Marine mammal Brucella genotype associated with zoonotic infection.

Marine Mammal Brucella Genotype Associated with Zoonotic Infection

A novel Brucella isolate in association with two cases of stillbirth in non-human primates - first report.

Background  Brucellosis is veterinary and human health problem.

Multiplex Assay Based on Single-Nucleotide Polymorphisms for Rapid Identification of Brucella Isolates at the Species Level†

ABSTRACT The genus Brucella includes a number of species that are major animal pathogens worldwide and significant causes of zoonotic infections of humans. Traditional methods of identifying Brucella to the species level can be time-consuming, can be subjective, and can pose a hazard to laboratory personnel in the absence of suitable biocontainment facilities. Using a robust phylogenetic framework, a number of single-nucleotide polymorphisms (SNPs) that define particular species within the genus were identified. These SNPs were used to develop a multiplex SNP detection assay, based on primer extension technology, that can rapidly and unambiguously identify an isolate as a member of one of the six classical Brucella species or as a member of the recently identified marine mammal group.

Brucella vulpis sp. nov., isolated from mandibular lymph nodes of red foxes (Vulpes vulpes).

Two slow-growing, Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains F60T and F965), isolated in Austria from mandibular lymph nodes of two red foxes (Vulpes vulpes), were subjected to a polyphasic taxonomic analysis. In a recent study, both isolates were assigned to the genus Brucella but could not be attributed to any of the existing species. Hence, we have analysed both strains in further detail to determine their exact taxonomic position and genetic relatedness to other members of the genus Brucella. The genome sizes of F60T and F965 were 3 236 779 and 3 237 765 bp, respectively. Each genome consisted of two chromosomes, with a DNA G+C content of 57.2 %. A genome-to-genome distance of >80 %, an average nucleotide identity (ANI) of 97 % and an average amino acid identity (AAI) of 98 % compared with the type species Brucella melitensis confirmed affiliation to the genus. Remarkably, 5 % of the entire genetic information of both strains was of non-Brucella origin, including as-yet uncharacterized bacteriophages and insertion sequences as well as ABC transporters and other genes of metabolic function from various soil-living bacteria. Core-genome-based phylogenetic reconstructions placed the novel species well separated from all hitherto-described species of the genus Brucella, forming a long-branched sister clade to the classical species of Brucella. In summary, based on phenotypic and molecular data, we conclude that strains F60T and F965 are members of a novel species of the genus Brucella, for which the name Brucella vulpis sp. nov. is proposed, with the type strain F60T ( = BCCN 09-2T = DSM 101715T).

Evaluation of MALDI-ToF as a method for the identification of bacteria in the veterinary diagnostic laboratory

Matrix-Assisted Laser Desorption Ionisation-Time of Flight (MALDI-ToF) Mass Spectrometry with Bruker MALDI Biotyper software was evaluated as a method for identifying veterinary bacteria. For 620 isolates (~100 bacterial species), identification by MALDI-ToF and non-16S rDNA methods (mainly phenotypic/biochemical) agreed to species-level (95.3%) and to species/genus-level (100%), but in the absence of 16S rDNA as a gold standard. For a further panel of 107 anaerobes and 234 aerobes (~100 bacteria species) using 16S rDNA results as the gold standard, MALDI-ToF/biochemical tests showed 97.8/96.6% species-level and 99.6/93.5% genus-level agreement for aerobes and 95.3/93.6% species-level and 100/95.3% genus-level agreement for anaerobes compared to the gold standard. Where results were obtained from direct spots, direct spots overlaid with formic acid and extracts, 89.4% of 180 aerobes and 90.1% of 152 anaerobes were identified by MALDI-ToF. MALDI-ToF was shown to be a rapid and reliable method to identify veterinary bacteria.

Extended Multilocus Sequence Analysis to Describe the Global Population Structure of the Genus Brucella: Phylogeography and Relationship to Biovars

An extended multilocus sequence analysis (MLSA) scheme applicable to the Brucella, an expanding genus that includes zoonotic pathogens that severely impact animal and human health across large parts of the globe, was developed. The scheme, which extends a previously described nine locus scheme by examining sequences at 21 independent genetic loci in order to increase discriminatory power, was applied to a globally and temporally diverse collection of over 500 isolates representing all 12 known Brucella species providing an expanded and detailed understanding of the population genetic structure of the group. Over 100 sequence types (STs) were identified and analysis of data provided insights into both the global evolutionary history of the genus, suggesting that early emerging Brucella abortus lineages might be confined to Africa while some later lineages have spread worldwide, and further evidence of the existence of lineages with restricted host or geographical ranges. The relationship between biovar, long used as a crude epidemiological marker, and genotype was also examined and showed decreasing congruence in the order Brucella suis > B. abortus > Brucella melitensis. Both the previously described nine locus scheme and the extended 21 locus scheme have been made available at http://pubmlst.org/brucella/ to allow the community to interrogate existing data and compare with newly generated data.

Isolation of a novel thermophilic Campylobacter from cases of spotty liver disease in laying hens and experimental reproduction of infection and microscopic pathology

The condition known as spotty liver disease or spotty liver syndrome can cause significant mortality in free range laying hen flocks. It has been described in Europe and Australia but the aetiology has not been established. There are similarities between spotty liver disease and avian vibrionic hepatitis, a condition which was reported in the 1950s. A Vibrio-like organism was suspected to be the cause of avian vibrionic hepatitis, although this organism was never fully characterised. We report the isolation of a novel Campylobacter from five separate outbreaks of spotty liver disease. The conditions required for culture, the growth characteristics, electron microscopical morphology and results of the phenotypic tests used in the identification of this novel Campylobacter sp. are described. The novel Campylobacter is slow growing and fastidious and does not grow on media routinely used for isolating Campylobacter sp. The morphology is typical for a Campylobacter sp. and phenotypic tests and a duplex real time PCR test differentiate the novel Campylobacter from other members of the genus. 16S rRNA analysis of 19 isolates showed an identical sequence which appears to represent a hitherto unknown sub lineage within the genus Campylobacter. Experimental intraperitoneal infection of four week old SPF chickens produced microscopic liver pathology indistinguishable from natural disease and the novel Campylobacter was recovered from the experimentally infected chicks. The isolates described appear to be a possible causal organism for spotty liver disease.

A novel taxon within the genus Actinobacillus isolated from alpaca (Vicugna pacos) in the United Kingdom

Members of the genus Actinobacillus comprise a diverse group of bacteria associated with mammals and birds including both pathogens and commensals. Here we describe the isolation of a previously undescribed Actinobacillus-like organism from seven epidemiologically unrelated infections of alpaca. The isolates are phenotypically and genotypically distinct from any previously described Actinobacillus species but 16S rRNA analysis unequivocally places the isolates as a novel lineage within the Actinobacillus sensu stricto. The clinical relevance of the organism requires further study however isolation in pure culture from organs of some cases suggests it may be associated with septicaemia in juvenile alpaca.

Isolation of Brucella ceti from a Long-finned Pilot Whale (Globicephala melas) and a Sowerby's Beaked Whale (Mesoploden bidens)

Abstract Brucella ceti is an emerging zoonotic pathogen that has been recovered from several species of cetaceans in the world’s oceans over the past 20 yr. We report the recovery of B. ceti from a Sowerby’s beaked whale (Mesoploden bidens) and a long-finned pilot whale (Globicehala melas). Recovery from the testis of a long-finned pilot whale provides further evidence of potential for B. ceti infection to impact the reproductive success of cetaceans, many of which are threatened species. The addition of another two cetacean species to the growing number from which B. ceti has been recovered also further emphasizes the concern for human infections with this organism.

HELICOBACTER CETORUM INFECTION IN STRIPED DOLPHIN (STENELLA COERULEOALBA), ATLANTIC WHITE-SIDED DOLPHIN (LAGENORHYNCHUS ACUTUS), AND SHORT-BEAKED COMMON DOLPHIN (DELPHINUS DELPHUS) FROM THE SOUTHWEST COAST OF ENGLAND

Abstract Helicobacter infection in cetaceans was first reported from the US in 2000 when the isolation of a novel Helicobacter species was described from two Atlantic white-sided dolphins (Lagenorhynchus acutus). Since then, Helicobacter species have been demonstrated in cetaceans and pinnipeds from around the world. Since 1990, the Animal Health and Veterinary Laboratories Agency Polwhele, Truro, has been involved in the UK Cetacean Strandings Investigation Programme to establish the cause of death of cetacean species stranded along the coast of Cornwall, England. We describe the isolation of Helicobacter cetorum in a striped dolphin (Stenella coeruleoalba) and evidence of H. cetorum infection in cetaceans from European waters.